(18) Assay Card

General Information
Summary Kalata B1, circulin A and B and cyclopsychotride shown to possess some anti-bacterial activity.
Condition A sensitive and reproducible two-stage radial diffusion assay was used for testing the antimicrobial activity of cyclic peptides. A 1-4 x 10^6 colony-forming unit/ml of the organism was mixed with 10 ml of molten underlay gel solution and was poured into 10- x 10-cm Petri dishes (Nunc) to form a uniform layer. The gel solution contained 10 mM sodium phosphate buffer, 0.03% TSB, and 0.02% Tween 20. One gel solution had high salt (100 mM NaCl) whereas the other did not. After the gel solidified, gel wells were made by a 3-mm diameter template in an evenly spaced array. An aliquot of 5 µl of a serial half-log dilution of testing peptides at seven concentrations was added to each well after removing the gel plugs. The dishes were incubated at 37oC for 3h to allow the peptides to diffuse into the underlay gels. Gels were overlaid with 10 ml of 1% agarose in 6% of TSB (wt/vol). After further incubation at 37oC for 16-24 h, the diameter of the clear zone surrounding the wells (colony-free) was measured under the microscope. Antimicrobial activities were expressed in units (0.1 mm =1 unit), and the MICs were determined from the x intercepts of the dose-response curves.
Result Kalata and Circulin A were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of 0.2 µM. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, Circulin B and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, Circulin B showed potent activity against E.coli with a minimum inhibitory concentration of 0.41 µM. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans.
AssayType Anti-bacterial

Tam JP, Lu YA, Yang JL, Chiu KW (1999) An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc Natl Acad Sci U S A 96:8913-8

Proteins Assayed circulin B
circulin A
kalata B1